Microbial biosurfactants as agents for controlling pests

ABSTRACT

Biosurfactants produced by microbes are used to control pests. The biosurfactants can be produced by cultivating a biosurfactant-producing microbe, producing a fermentation broth, and obtaining the biosurfactant from the fermentation broth. Alternately, the biosurfactants can be produced in situ in the environment of the pests by applying a carbon substrate to the pests&#39; environment, which permits naturally-occurring biosurfactant-producing microbes to grow and to generate the biosurfactants. The biosurfactants have pesticidal qualities, and can be used to control a variety of pests, while being biodegradable and otherwise avoiding adverse environmental effects that have often been associated with conventional synthetic pesticides.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/575,913, filed Jun. 1, 2004, and of U.S.Provisional Patent Application Ser. No. 60/604,139, filed Aug. 23, 2004,which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The invention relates to methods and compositions for the control ofpests. More specifically, the present invention relate to microbialbiosurfactants and biosurfactant-producing microbes used as agents forcontrolling pests.

2. The Relevant Technology

Although chemical pesticides are valuable in the control of pests, theiruse poses many problems. They tend to harm non-target organisms such ashumans, domestic animals, beneficial insects, and wildlife. In addition,their residues tend to remain on the crop and may accumulate in thesoil, water, or air. Another concern is the development of resistance topesticides by the targeted organisms. Due to the serious environmentalproblems associated with chemical pesticides, the demand for saferpesticides and alternate pest control strategies is increasing.

It has become widely known in the art that certain live biologicalcontrol agents (bacteria, phage, bacteriophages) have some use in pestcontrol. The success of these biological control agents depends onunderstanding the biology of these agents and of the target pest.Several limitations are associated with the use of these biologicalagents according to conventional techniques. One of the limitations isthat some of the biologic control agents are opportunistic pathogensagainst humans or animals. Another constraint is the competition betweenthe native organisms (beneficial or pathogens) in the ecosystem and thecontrol agents. In many cases, the control agent is not able to competein the environment and the efficacy of the treatment is questionable.

U.S. Pat. No. 5,767,090 describes the use of rhamnolipids against thetransitional zoosporic stage of zoosporic fungi. Although rhamnolipidsare very effective against this motile stage of the pathogenic fungi,successful treatment in commercial crops production depends greatly onapplication time and repetitive applications are sometimes necessary toachieve the desired control. During wet conditions foliar applicationmay be impractical and repetitive applications significantly increasesproduction cost.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods and compositions, based uponnatural microbial biosurfactants, for controlling pests. Examples of thepests treated with the biocontrol agents include insects such as ants,aphids, thrips, whiteflies, scales, lice, cockroaches, termites,houseflies, mosquitoes, etc; arachnids such as mites, spiders, ticks;nematodes; mollusks; amoeba; parasites; and algae. The agents forcontrolling pests according to the invention include natural pesticides(biopesticides) derived of microbial origin. Biopesticides areinherently less harmful than conventional pesticides. They oftenbiodegrade quickly, therefore resulting in low exposures and largelyavoiding the pollution problems caused by conventional pesticides.

Embodiments of the invention extend to the use of naturally occurringmicrobial biosurfactant compounds to control pests. The invention alsoextends to methods of controlling pests using biosurfactant-producingmicroorganisms. The invention further encompasses the compositionsthemselves, as well as the methods for manufacturing or producing thecompositions and their application to pest control as disclosed herein.Other objects of the invention may be apparent to one of skill in theart upon learning of the invention described herein.

In another embodiment of the invention, formulations and treatments tocontrol pests are achieved by the addition of carbon, or organic,substrates, such as oil or fatty acids, to target areas (before orduring infestation) to support the growth of biosurfactant-producingorganisms as well as to produce the desired biosurfactants on site toachieve the objective of the invention. Natural biosurfactant-producingorganisms at the site of application (soil, aquatic system, plant parts,etc) will produce biosurfactants while utilizing the carbon substrate.During this process, the biosurfactant produced will destroy or paralyzepests in the targeted area. According to the invention, it is notnecessary to pre-inoculate the targeted site or the carbon substratemixture with biosurfactant-producing organisms.

Microbial biosurfactants are compounds produced by variety ofmicroorganisms such as bacteria, fungi, and yeasts. Biosurfactants forman important class of secondary metabolites that occur in manymicroorganisms such as Pseudomonas species (P. aeruginosa, P. putida, P.florescens, P. fragi, P. syringae); Flavobacterium spp.; Bacillus spp.(B. subtilis, B. pumillus, B. cereus, B. licheniformis); Candida species(C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C. torulopsis);Rhodococcus sp.; Arthrobacter spp.; campylobacter spp.; cornybacteriumspp. and so on.

Biosurfactants are biodegradable and can be easily and cheaply producedusing selected organisms on renewable substrates. Mostbiosurfactant-producing organisms produce biosurfactants in response tothe presence of hydrocarbon source (e.g. oils, sugar, glycerol, etc) inthe growing media. Other media components can also affect biosurfactantproduction significantly. For example, the production of rhamnolipids bythe bacteria Pseudomonas aeruginosa can be increased if nitrate is usedas a source of nitrogen rather than ammonium. Also the concentration ofiron, magnesium, sodium, and potassium; the carbon:phosphorus ratio, andagitation greatly affect rhamnolipid production.

Biosurfactants include low-molecular-weight glycolipids (GLs),lipopeptides (LPs), flavolipids (FLs), phospholipids, andhigh-molecular-weight polymers such as lipoproteins,lipopolysaccharide-protein complexes, and polysaccharide-protein-fattyacid complexes. Biosurfactants have a great deal of structuraldiversity. The common lipophilic moiety of a biosurfactant molecule isthe hydrocarbon chain of a fatty acid, whereas the hydrophilic part isformed by ester or alcohol groups of neutral lipids, by the carboxylategroup of fatty acids or amino acids (or peptides), organic acid in thecase of flavolipids, or, in the case of glycolipids, by thecarbohydrate.

According to the embodiments of the invention, these compounds may alsobe synthesized by standard organic synthesis methods.

In one embodiment, a single biosurfactant or a mixture of differentbiosurfactants may be used in a formulation to perform the functions andachieve the results disclosed herein.

Co-pending U.S. Provisional Patent Application Ser. No. 60/604,139,filed Aug. 23, 2004, is incorporated herein by reference and describesbiosurfactants (e.g. GLs, FLs, and LPs etc) having a powerfulpenetrating capabilities. It has been discovered that an importantcharacteristic of these biosurfactants or biopesticides is that they areable to penetrate tissues or cells. This is a very important factor thatinfluences effectiveness of pesticides. In general, the effectiveness ofpesticides can be significantly enhanced if they are able to readilyspread on the treated surface and to penetrate into the pest (e.g., intothe insects' cuticle). According to embodiments of this invention, thebiopesticide is able to penetrate through pests' tissues sufficientlyand to be effective in lesser amounts without the use of adjuvants. Ithas been found that at concentrations above the critical micelleconcentration, the biosurfactant are able to penetrate more effectivelyinto treated objects.

In this invention, we have unexpectedly discovered that some natural andnon-toxic biosurfactants such as rhamnolipids have unique mode of actionagainst some pests and parasites and this broad-spectrum activitydiffers from conventional pesticides mode of action.

The development of the natural biopesticides of the invention, which canbe produced in high amounts using selected microorganisms for pestcontrol, represents a significant advancement in the art. As notedabove, pests can be controlled using either the biosurfactant-producingorganisms as a biocontrol agent or by the biosurfactants themselves. Inaddition, pests control can be achieved by the use of specificsubstrates to support the growth of biosurfactant-producing organisms aswell as to produce biosurfactants pesticidal agents. Thus, according tothe invention, the microbial biosurfactants' glycolipids, such asrhamnolipids, sophorlipids, trehalose lipids; flavolipids; lipopeptides;etc., as pesticidal agents have the potential to reduce the need for anduse of synthetic pesticides.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other aspects of the present invention,a more particular description of the invention will be rendered byreference to specific embodiments thereof which are illustrated in theappended drawings. It is appreciated that these drawings depict onlytypical embodiments of the invention and are therefore not to beconsidered limiting of its scope. The drawings are not drawn to scale.The invention will be described and explained with additionalspecificity and detail through the use of the accompanying drawings inwhich:

FIG. 1 is a flow diagram illustrating a method of applying microbialbiosurfactants to control pests according to an embodiment of theinvention.

FIG. 2 is a flow diagram illustrating a method of obtainingbiosurfactants in a concentration that is sufficient to be used tocontrol pests.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention is directed to compositions and methods forcontrolling pests, and, more particularly, to pesticides derived fromnatural substances, such as microbial or fermentation metabolites.According to embodiments of the invention, the produced fermentationbroth containing the microbial biosurfactant may be used withoutextraction or purification. If desired, extraction and purification ofthe biosurfactants can be easily achieved using standard extractionmethods or techniques described in the literature.

In one aspect of the invention, fermentation broth or the purifiedbiosurfactants, e.g., GLs, FLs, LPs, etc., may be used to protect cropplants, homes, structures, soils, aquatic systems, ponds, fishaquariums, humans, or animals by controlling harmful pests. As usedherein, the term “control” used in reference to the activity produced bythe biosurfactants or biosurfactant-producing organisms extends to theact of killing, disabling or immobilizing pests or otherwise renderingthe pests substantially incapable of causing harm.

In another aspect of the invention, biosurfactant-producing organisms,e.g., Pseudomonas spp. may be added to the soil, plants' growing medium,plants, aquatic medium, or any area to be treated. The organisms cangrow onsite and produce the biosurfactants to control the pests targetedas described in this invention. The cultures may be mixed with growthenhancement substances to aid in their growth and the production of themicrobial biosurfactants. Substances such as oil, glycerol, sugar, orother nutrients may also be used.

In another embodiment of the invention, carbon substrate to support thegrowth of biosurfactant producing organisms is added to the pestinfested areas, soil, plants' growing medium, plant parts, aquaticmedium, or any area to be treated. Biosurfactant producing organisms cangrow on the substrate to produce biosurfactant in place and control thetargeted pests as described in this invention. It is not necessary toadd biosurfactant-producing organisms to the substrate. Naturalbiosurfactant producing organisms found at the site of application willbe able to grow and produce the biosurfactant. Examples of carbonsubstrates that can be added to the targeted areas include, but notlimited to, organic carbon sources such as natural or synthetic oilincluding used frying oil; fat; lipid; wax (natural or paraffin); fattyacids such as lauric; myristic, etc; fatty acid alcohol such as laurylalcohol; amphiphilic esters of fatty acids with glycerol such asglyceryl monolaurate; glycol esters of fatty acid such as polyethylenemonostearate; fatty acid amines such as lauryl amine; fatty acid amides;hexanes; glycerol; glucose; etc. It is preferable to use water insolublecarbon substrate to encourage excessive production of thebiosurfactants. In addition to the carbon substrate, nutrients such asvitamins, inorganic minerals may also be added to the substrate toencourage biosurfactant-producing organisms growth. Although it is notnecessary, it is preferable to spike or amend the carbon substrate witha sufficient amount of specific biosurfactant to initiate theemulsification process and to inhibit or reduce the growth of othercompeting organisms for the biosurfactant-producing organism and tocontrol pests. An illustrative but not restrictive example would be theaddition of 60-100 mg/l rhamnolipid biosurfactant in the final dilutedoil substrate mixture. The diluted mixture is applied to the area to betreated. This method aids in growth establishment of Pseudomonasaeruginosa or florescens (rhamnolipid producing organisms) populationand reduces the chance of growth of its competing or disease causingorganisms, the Phytophthora, nematodes, Bacillus sp. If it is desired toproduce Syringotoxin lipopeptide biosurfactants, a small amount ofsyringotoxins (less than few mg/l) is added to the oil-glycerolsubstrate. Syringotoxin will eliminate many competing organisms andmaintain Pseudomonas syringae growth while producing the lipopeptidetoxins. Pseudomonas syringae and Bacillus subtilis for instance produceseries of lipopeptides biosurfactants referred to as porens. Theselipopeptide porens include pseudomycin, syringomycin, tabtoxin,phaseolotoxin, and surfactin. Some lipopeptides are capable of creatingholes in cell membranes, cells, and tissues. Due to their powerfulactivity on cells and tissues, these biosurfactants are very useful incontrolling algae, nematodes, insects and other pests. Pseudomycin canbe applied as a pre-plant treatment for nematode or insects larvaecontrol in crop production. If it is desired to encourage the growth ofBacillus subtilis, a small amount of surfactin biosurfactant is added tothe carbon substrate medium to aid in establishment of subtilispopulation and the production of more surfactin on-site.

The use of carbon substrates to produce biosurfactants at the targetedsites especially in the presence of minute amount of biosurfactant as abiocontrol starting point, greatly enhances the efficacy of thetreatment, broadens the biocontrol spectrum against many pathogens, andreduces the frequency and cost of application of the biosurfactant. Asit will be described in the examples section, this is very essential forsoil treatment applications.

Synthetic surfactants such as alkyl betaines e.g. Lauryl betaine, alkylsulfates as lauryl sulfate or its salt, alkyl ammonium bromidederivatives, alkyl phenol ethoxylates, alkyl ethylene (or polyethylene)ethoxylates may be used to lower the surface tension and facilitate theutilization of the carbon substrate by the naturalbiosurfactant-producing organisms but it is preferable to use naturalbiosurfactants that are able to inhibit the growth of competingorganisms and enhance the growth of the specific biosurfactant producingorganisms as described in the invention.

Derivatives of these microbial biopesticides or compounds with similarstructures or characteristics and able to control pests as alsodisclosed herein and are encompassed by embodiments of the invention.

It has been observed that some of the mentioned synthetic surfactantsabove may have inhibitory effects against some pathogens and may also beused as active agents to control pests such as insects, algae, parasiticamoeba, nematodes, weeds or other pests as described in this invention.They may also be used in conjunction with the natural biosurfactants.

According to this invention, biosurfactants (e.g. GLs, FLs, and LPs etc)have a powerful biopesticidal activity against many pests and diseasesaffecting plants and these biosurfactants also have similar biopesticideactivity against pests and diseases affecting humans and animals. Pestscontrolled include insects, their larvae and eggs; mites; algae(seaweeds, pond algae, and the microscopic algae such as blue-greenalgae); microbial pests (nematodes, bacteria, fungi, parasites, amoeba,protozoa, viruses, etc); mollusks; worms; and plant weeds. In addition,these biosurfactants may be used to treat human diseases such asova-parasites and cysts, hair dandruff, etc. In addition, rhamnolipidbiosurfactant is an effective spennicide at a concentration of 250 ppm.Examples of animal diseases include, but not limited to, dog's heartworm; fish parasites and microbial infections such as whirling diseasecaused by the amoeba Myxobolus, fish fungal disease (water mold) orgreen algae; fish protozoa disease such as Chilodonella; fish parasitesas gill and skin flukes. Also cattle hoof diseases can also becontrolled as described in this invention. Animals are treated bydipping or bathing in a biosurfactant solution alone or in the presenceof other compounds such as copper or zinc.

The natural biosurfactants' active components may be used according tothe invention either alone or combined with other acceptable active orinactive (inert) components that may be used as adjuvants or may havepesticidal activity. It is preferable to use adjuvants or pesticidalcomponents of natural source to complement the natural aspects of thebiosurfactants. These components can be either an oil component such ascinnamon oil, clove oil, cottonseed oil, garlic oil, or rosemary oil;another natural surfactant such as Yucca or Quillaja saponins; or thecomponent may be an aldehyde such as cinnamic aldehyde. Other oils thatmay be used as a pesticidal component or adjuvants include: almond oil,camphor oil, castor oil, cedar oil, citronella oil, citrus oil, coconutoil, corn oil, eucalyptus oil, fish oil, geranium oil, lecithin, lemongrass oil, linseed oil, mineral oil, mint or peppermint oil, olive oil,pine oil, rapeseed oil, safflower oil, sage oils, sesame seed oil, sweetorange oil, thyme oil, vegetable oil, and wintergreen oil. Othersuitable additives, which may be contained in the formulations accordingto the invention, are all substances, which are customarily used forsuch preparations. Example of such additives include adjuvants,surfactants, emulsifying agents, plant nutrients, fillers, plasticizers,lubricants, glidants, colorants, pigments, bittering agents, bufferingagents, solubility controlling agents, pH adjusting agents,preservatives, stabilizers and ultra-violet light resistant agents.Stiffening or hardening agents may also be incorporated to strengthenthe formulations and make them strong enough to resist pressure or forcein certain applications such as soil, root flare or tree injectiontablets.

Examples of buffering agents include organic and amino acids or theirsalts. Suitable buffers include citrate, gluconate, tartarate, malate,acetate, lactate, oxalate, aspartate, malonate, glucoheptonate,pyruvate, galactarate, glucarate, tartronate, glutamate, glycine,lysine, glutamine, methionine, cysteine, arginine and a mixture thereof.Phosphoric and phosphorous acids or their salts may also be used.Synthetic buffers are suitable to be used but it is preferable to usenatural buffers such as organic and amino acids or their salts listedabove.

Examples of solubility control agents or excipients may be used in theformulations to control the release of the active substances may includewax, chitin, chitosan, C12-C20 fatty acids such as myristic acid,stearic acid, palmitic acid; C12-C20 alcohols such as lauryl alcohol,cetyl alcohol, myristyl alcohol, and stearyl alcohol; amphiphilic estersof fatty acids with glycerol, especially monoesters C12-C20 fatty acidssuch as glyceryl monolaurate, glyceryl monopalmitate; glycol esters offatty acids such as polyethylene monostearate orpolypropylenemonopalmitate glycols; C12-C20 amines such as lauryl amine,myristyl amine, stearyl amine, and amides C12-C20 fatty acids.Additionally, the solubility control agent can be a swellable polymer,such as a crosslinked swellable polyacrylamide, in order to control therelease of the active substances.

Examples of pH adjusting agents include Potassium hydroxide, ammoniumhydroxide, Potassium carbonate or bicarbonate, hydrochloric acid, nitricacid, sulfuric acid or a mixture.

Additional components such as an aqueous preparation of a salt aspolyprotic acid such as sodium bicarbonate or carbonate, sodium sulfate,sodium phosphate, sodium biphosphate, can be included in theformulation.

According to embodiments of this invention, the microbial biopesticidescan be produced and formulated in a variety of ways, including liquid,solids, granular, dust, or slow release products by means that will beunderstood by those of skill in the art upon learning of the inventiondisclosed herein.

They may be applied by spraying, pouring, dipping, in the form ofconcentrated or diluted liquids, solutions, suspensions, powders, andthe like, containing such concentrations of the active agent as is mostsuited for a particular purpose at hand. They may be applied as is orreconstituted prior to use. For example, they may be applied by directinjection into trees or root flares.

Solid formulations of the invention may have different forms and shapessuch as cylinders, rods, blocks, capsules, tablets, pills, pellets,strips, spikes, etc. Solid formulations may also be milled, granulatedor powdered. The granulated or powdered material may be pressed intotablets or used to fill pre-manufactured gelatin capsules or shells.Semi solid formulations can be prepared in paste, wax, gel, or creampreparations.

For human or animal applications, the formulations may be prepared inliquid, paste, ointment, suppository, capsule or tablet forms and usedin a way similar to drugs used in the medicinal drugs industry. Theformulations can be encapsulated using components known in thepharmaceutical industry. Encapsulation protects the components fromundesirable reactions and helps the ingredients resist adverseconditions in the environment or the treated object or body e.g.stomach.

The compositions according to the invention can be applied to theplants, pests, or soil using various methods of application. Each methodof application may be preferred under certain circumstances.

The compositions according to the invention may be used to introduce theactive compounds into the soil. These preparations could be incorporatedinto the soil in the vicinity of the roots of the plants. This could bein the form of liquid, bait, powder, dusting, or granules, or they areinserted in the soil as tablets, spikes, rods, or other shaped moldings.

The compositions according to the invention can be used for treatingindividual trees or plants. For example, the formulations can be moldedin different shapes or forms (solid, paste or gel, or liquid) andintroduced into the vascular tissue of the plants. Moldings forms can beas tablets, capsules, plugs, rods, spikes, films, strips, nails, orplates. The shaped moldings can be introduced into pre-drilled holesinto the plants or root flares, or they can be pushed or punched intothe cambium layer.

Another method of application of the invention is the use of dispensingdevices such as syringes, pumps or caulk guns, paste-tubes or plungertubes for delivering semi-solid formulations (paste, gel, cream) intodrilled holes in tree trunks or root flares.

The compositions of the invention can be applied in the form of paste,gel, coatings, strips, or plasters onto the surface of the plant. In onemethod, a plaster or strip may have the semi-solid formulation, e.g.,insecticide placed on the side that will contact the tree, bush, or roseduring the treatment. The same strip may have glue or adhesive at one orboth ends to wrap around or stick to the subject being treated.

The compositions according to the invention can be sprayed or dusted onthe leaves in the form of pellets, spray solution, granules, or dust.

The solid or semi-solid compositions of the invention can be coatedusing film-coating compounds used in the pharmaceutical industry such aspolyethylene glycol, gelatin, sorbitol, gum, sugar or polyvinyl alcohol.This is particularly essential for tablets or capsules used in pesticideformulations. Film coating can protect the handler from coming in directcontact with the active ingredient in the formulations. In addition, abittering agent such as denatonium benzoate or quassin may also beincorporated in the pesticidal formulations, the coating or both.

The compositions of the invention can also be prepared in powderformulations and filled into pre-manufactured gelatin capsules.

The concentrations of the ingredients in the formulations andapplication rate of the compositions may be varied widely depending onthe pest, plant or area treated, or method of application. As describedin greater detail below, the compositions and methods of the inventioncan be used to control a variety of pests, including insects and otherinvertebrates, algae, microbial pests, and, in some situations, weeds orother plants.

A purified mixture of rhamnolipids (supplied by Jeneil Biotech ofSaukville, Wis.) and Pseudomonas spp. fermentation broth filtrate wastested for their activity on different pests such as thrips, aphids,houseflies, mosquitoes, box-elder bugs, nematodes, spider mites andalgae. The cultured material was effective at a concentration as low as0.005% on some pests. The following are examples to illustrate theprocedures of practicing the invention. These examples are illustrativeand should not be construed as limiting.

EXAMPLES

Fermentation broth containing rhamnolipids was tested for itseffectiveness as insecticide to control houseflies. Ten houseflies wereconfined in petri dishes covered with screen through which theinsecticide was sprayed. Another set of ten houseflies were sprayed withwater and kept as control. Table 1 shows the results of the test.

TABLE 1 Effect of rhamnolipid treatments on housefly survival.Rhamnolipid Treatments 0% 3% Time(min.) Houseflies Survival Rate 2 100100 5 100 90 7.5 100 70 10 100 30 12 100 20 15 100 0

In another test, purified rhamnolipid material at 2.5% concentration wassprayed directly on spiders in naturally infested area. Treatmentincluded six spiders sprayed with the pesticide as a test, while sixspiders were sprayed with water as control. Full control of the spidermites was achieved in less than fifteen minutes after treatment.

A naturally infested lemon tree with spider mites was sprayed with a1.25% rhamnolipid solution. The mites were observed using magnifyingglass for movement. Death was noted in less than 15 minutes.

Naturally infested tomato plants with whiteflies were sprayed with 0.1%rhamnolipid mixture diluted in water. Control plants were sprayed withwater only. Whiteflies sprayed with the rhamolipids stuck to the leavesand weren't able to move after the treatment. Full control was achievedin less than 6 minutes.

Into each 1-liter water bottle a tablet containing 0, 0.075, or 0.2grams rhamnolipid (put example) was added. Ten-mosquitoes larvae weretransferred into each of the bottles. Total death of the larva wasobserved in about 2 hours and 40 minutes in the bottle containing 0.2grams rhamnolipid. In the bottle containing 0.075 grams rhamnolipid,only one live mosquito larva was left after 24 hours of studyinitiation. No death was observed in the control treatment. Thissignificant discovery is critical in the control and spread of the WestNile Virus Vector. In another study we found that rhamnolipid additionat concentration of 100 ppm prevented mosquitoes eggs from hatching.

A Petri dish containing fifty ml of water infested with amoeba wastreated with 250 ppm rhamnolipid. Examining the amoeba under themicroscope before and after the treatment showed that within fiveminutes of rhamnolipid addition that the amoeba collapsed anddisintegrated.

An infested red ants mound was drenched with 0.5% rhamnolipid solution.The treatment was effective and the mound was free of ants for more than2 weeks.

Pesticidal preparations:

Example A: A 5% rhamnolipid solution was prepared using 25% purifiedrhamnolipid concentrate supplied by Jeneil Biotech.

Example B: A concentrated solution was prepared by mixing 20 gramssesame oil, 30 grams canola oil, 10 grams glycerol, and 40 grams water.The mixture is diluted 10, 50 or 100 times with water prior to use.

Example C: A concentrated solution was prepared by mixing 5 gramsrhamnolipids, 20 grams sesame oil, 30 grams canola oil, 10 gramsglycerol, and 35 grams water. The mixture was diluted 10, 50, or 100times with water prior to use.

Example D: Water used as a control treatment.

Example E: A concentrated Solution of 10% phosphite and 8% potassium wasprepared by mixing 70% phosphorous acid, 45% potassium hydroxide, andwater. The solution was buffered with citrate/gluconic acid to pH of5.8. The mixture was diluted 100 times with water prior to use.

Example F: A concentrated Solution of 5% rhamnolipid, 10% phosphite and8% potassium was prepared by mixing 70% phosphorous acid, 45% potassiumhydroxide, and water. The solution was buffered with citrate/gluconicacid to pH of 5.8. The mixture was diluted 100 times with water prior touse.

Grasshopper Study:

For each treatment, six grasshoppers were sprayed with solutionsprepared from Examples A, B, C, or D diluted 10 or 100 times with water.At ten times dilution, Example C treatment was the most effective andkilled all the treated grasshoppers within ten minutes. Example Atreatment at 10 times dilution instantly slowed down the movement of thegrasshoppers, but half of the treated recovered within 20 minutes ofapplication. Example B treatment at 10 times dilution had similar effectlike that of rhamnolipid alone. At 100 times dilution, example A was noteffective. Example B treatment was 33% effective and example C treatmentwas 84% effective. Water treatments had no effect on grasshoppers.

Powdery Mildew Study:

Diluted solutions of Examples A, B, C, or D were sprayed on squash androses plants heavily infected with Powdery mildew. Results of rosetreatments are presented in Table 2. It was interesting to note thatupon spraying the roses with example C formulation at 100 timesdilution, the infected area washed out completely from the leaves. Theresults of squash treatments were similar to rose treatments, but squashplants were more sensitive to the spray solutions. At 10 times dilution,squash leaves developed necrotic tissues within 24 hours of the sprayapplication and the plants shut down and died within three days.Examples E and F were tested on roses only. An important finding inpowdery mildew treatments is that neither rhamnolipid nor phosphitealone was very effective against the powdery mildew at theconcentrations used; however, the rhamnolipid/phosphite combination wasvery effective in the treatment of powdery mildew disease. Althoughpowdery mildew does not belong to the zoosporic fungi group, it isbelieved that the rhamnolipid enhances the activity and mode of actionof phosphite through membrane disturbance or by penetrating the fungusprotective layers.

TABLE 2 Effect of different formulations on the control of powderymildew on roses. DILU- DISEASE TION SEVER- PHYTO- TREATMENT FACTOR ITY*CONTROL TOXICITY** EXAMPLE A 10× 4 Limited 2 suppression 50× 5 No Effect1 100×  5 No Effect 1 EXAMPLE B 10× 2 Effective 4 suppression Total leafburn 50× 4 Limited 3 Suppression 100×  4 Limited 2 Suppression Localizedspots EXAMPLE C 10× 2 Effective 4 suppression 50× 1 Effective 2suppression 100×  1 Very effective 1 control EXAMPLE D Water 1 No Effect1 CONTROL EXAMPLE E 100×  5 No Effect 1 EXAMPLE F 100×  2 Suppressionfor 1 more than 10 days *Treated plants were visually examined fordisease symptoms on the leaves. Evaluation was documented on scale of1-5, where 1 = No Powdery Mildew, 2 = 1-25% infection, 3 = 26-50%infection, 4 = 51-75% infection, 5 = 76-100% infection (all the leavesare infected). Phyto-toxicity was documented on scale of 1-4 where 1 =No necrosis, 2 = 25% of leaf is necrotic, 3 = 50% leaf damage, and 4 =total leaf damage.Herbicidal Activity:

Due to the powerful micro-emulsifying and penetrating activity of thebiosurfactants, especially in combination with oil, they can be used asnonselective herbicides to control weed pests. At concentrations of 0.5%rhamnolipid and higher, necrosis was observed on some plants. Thiseffect is extremely magnified in the presence of oil especially sesameor cottonseed oil. At a concentration of 0.05% rhamnolipid and 2% oil,many treated weeds or plants were destroyed within few days of thetreatment.

Nematodes treatment:

Preliminary tests on nematodes were conducted according to the followingprocedure. The soil used in this test was isolated from a potato fieldnaturally infested with nematodes. Seventy-five grams of soil (15%initial water content) were wrapped in double folded piece ofcheesecloth and fitted in a strainer. The strainer containing the soilwas gently suspended in a plastic funnel containing 450 ml water(control), or 0.75% Rhamnolipid mixture. The bottom surface of thestrainer containing the soil was maintained in contact with thetreatment solutions throughout the study. Twenty-five ml samples werecollected at different times through the clamped tubing connected to thestem of the funnel. The supernatant solutions were directly transferredto a petri dish for examination using a microscope. The number ofnematode pests surviving was recorded at 24 hrs intervals for a periodof seven days. Mortality was concluded if individual nematodes areimmobile and fail to respond to disturbance with an eyelash cemented toa needle. The test was done in three replicates. The results arepresented in Table 3.

TABLE 3 Effect of rhamnolipid mixture treatments on nematodes control.Rhamnolipid Treatment 0% 0.75% Time (hrs) Total Nematode Control Rate(%) 24 7.7 16.3 48 23.1 35.4 72 27.7 48.3 96 34.6 60.5 120 39.2 74.8 14447.7 93.0

Different formulations and concentrations have been tested for activityagainst potato, tomato, and sugar beet nematodes and other pests.

After the nematode experiment was terminated, a surprise finding wasobserved on the nematodes treatment solutions present in the plasticfunnel. It was observed that the control (water only) solution supportedthe growth of algae after it was left in the sun for few weeks. On thecontrary, rhamnolipid treatment maintained clear solution with no algaegrowth. To verify the results, another set of treatments at 0 (water ascontrol), 0.005, 0.01, 0.1 and 1% rhamnolipid concentrations wereconducted. The water used in the experiment was collected from an algaeinfested pond. All rhamnolipid treatments did not support the growth ofalgae. However, at the lowest concentration of rhamnolipid, algae growthwas re-established after 6 weeks of the initiation of the study. Theother treatments were clean of algae during the three months study.Control treatment (pond water) turned greenish in color and the algaeflourished in the water.

Root Knot Eggs Nematode Study:

A set of nematode eggs taken from the roots of tomato plant infestedwith nematode galls were transferred into petri dishes containing either25 ml water or 250 ppm rhamnolipid in 25 ml water. The eggs wereperiodically examined under the microscope. Rhamnolipid treated eggs'color changed to brownish color during the course of the study and theeggs collapsed and disintegrated after 7 days. No change in eggs' coloror shape was observed in the water treatment.

Gel preparation: A 5% rhamnolipid Gel formulation is prepared byimpregnating 1.0% gum or carboxyvinyl carbopol polymer with purifiedrhamnolipid dissolved in water. The material is mixed using a vortex toyield a paste in less than 30 minutes. This treatment can be used to rubon animals for Ticks treatment.

FIG. 1 is a flow diagram that illustrates an example of the methods ofthe invention for controlling pests. The method begins by obtaining amicrobial biosurfactant (102). As described herein, the biosurfactantcan be obtained by a manufacturing or cultivation process that occursprior to applying the biosurfactant (104). Alternately, thebiosurfactant can be obtained by applying a carbon substrate to theenvironment of the pests (106) and permitting naturally-occurringmicrobes to grow on the substrate (108) and to thereby produce thebiosurfactant. In either case, the biosurfactant is applied to the pestsor to the environment of the pests (110), such that the pests aresubstantially controlled.

FIG. 2 is a flow diagram that illustrates an example of the methods ofthe invention for producing biosurfactants that can be used to controlpests. The method begins by cultivating a biosurfactant-producingmicrobe, including producing a fermentation broth containing thebiosurfactant (202). The biosurfactant is then obtained (204) from thefermentation broth in a concentration that can be applied to pests or toan environment in which the pests are located in an amount such that thepests are substantially controlled. Obtaining the biosurfactant from thefermentation broth can be performed in one of a variety of waysillustrated in FIG. 2. For instance, in certain embodiments, thefermentation broth includes the biosurfactants at a suitableconcentration (206) without requiring purification or extraction.Alternately, the fermentation broth can be purified (208) or thebiosurfactant can be extracted from the fermentation broth (210).Although these exemplary methods illustrated in FIG. 2 are suitable forobtaining biosurfactants, the methods of controlling pests disclosedherein can be performed regardless of the methods used to obtain thebiosurfactants.

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive.

The processes, methods of use and examples of components listed in theinvention are illustrative and not inclusive. The invention may beembodied in other specific forms without departing from its spirit oressential characteristics. The described embodiments are to beconsidered in all respects only as illustrative and not restrictive. Theappended claims are presented to illustrate the embodiments of theinvention disclosed herein.

1. A method for controlling pests, comprising: obtaining a microbial biosurfactant having a glycolipid and/or rhamnolipid; and providing the microbial biosurfactant to one or more pests in an amount such that the one or more pests are controlled, wherein the pests are nematodes.
 2. The method as defined in claim 1, wherein obtaining a biosurfactant comprises cultivating a microbe that produces the biosurfactant.
 3. The method as defined in claim 2, wherein cultivating the microbe comprises producing a fermentation broth containing the biosurfactant.
 4. The method as defined in claim 3, wherein the biosurfactant is obtained without purifying the fermentation broth or extracting the biosurfactant from the fermentation broth.
 5. The method as defined in claim 3, wherein cultivating the microbe further comprises extracting the biosurfactant from the fermentation broth.
 6. The method as defined in claim 3, wherein cultivating the microbe further comprises purifying the fermentation broth.
 7. The method as defined in claim 1, wherein the microbial biosurfactant has been produced by a microbe in the group that consists of: pseudomonas species, including P. aeruginosa, P. putida, P. florescens, P. fragi, and P. syringae; flavobacterium species; candida species, including C. albicans, C. rugosa, C. tropicalis, C. lipolytica, and C. torulopsis; rhodococcus species; arthrobacter species; campylobacter species; and cornybacterium species.
 8. The method as defined in claim 1, wherein the microbial biosurfactant is a penetrant that penetrates the pest without the use of a separate adjuvant.
 9. A method as defined in claim 1, wherein the microbial biosurfactant is combined with a solubility control agent.
 10. A method as defined in claim 9, wherein the solubility control agent is a cross-linked swellable polyacrylamide.
 11. A method as defined in claim 1, wherein the biosurfactant is combined with an oil.
 12. A method as defined in claim 1, wherein the biosurfactant is in a formulation having a form selected from the group consisting of liquid, solutions, suspensions, powders, pastes, waxes, gel, cream, cylinders, rods, blocks, capsules, tablets, pills, pellets, strips, or spikes.
 13. A method as defined in claim 12, wherein the formulation is applied to an ant mound.
 14. A method as in claim 1, wherein the providing is by applying the microbial biosurfactant to the one or more pests, soil, an aquatic system, a plant, an object surface, a home, a structure, a pond, or combination thereof.
 15. A method as in claim 1, wherein the controlled one or more pests are killed or paralyzed. 